Pediatric Pulmonology 43:20–28 (2008) Original Articles Differential Effects of Chronic Intermittent and Chronic Constant Hypoxia on Postnatal Growth and Development Reza Farahani,1,2,3 Amjad Kanaan,3,4 Orit Gavrialov,3,4 Steven Brunnert,5 Robert M. Douglas,3,4 Patrick Morcillo,3 and Gabriel G. Haddad, MD3,4,6* Summary. Exposure to chronic constant or intermittent hypoxia (CCH or CIH) may have different effects on growth and development in early life. In this work, we exposed postnatal day 2 (P2) CD1 mice to CCH or CIH (11% O2) for 4 weeks and examined the effect of hypoxia on body and organ growth until P30. Regression analysis showed that weight increased in control, CCH and CIH cohorts with age with r2 values of 0.99, 0.97, and 0.94, respectively. Between days 2 and 30, slopes were 0.93
0.057, 0.76
0.108, and 0.63
0.061 (g/day, means
SEM) for control, CIH, and CCH, respectively and significantly different from each other (P < 0.001). The slopes between P2 and P16 were 0.78
0.012, 0.46
0.002, and 0.47
0.019 for control, CCH and CIH, respectively. From P16 to 30, slopes were 1.12
0.033, 1.09
0.143, and 0.82
0.08 for control, CIH, and CCH, respectively with no significant difference from each other, suggesting a catch-up growth in the latter part of the hypoxic period. Slower weight gain resulted in a 12% and 23% lower body weight in CIH and CCH mice (P < 0.001) by P30. Lung/body ratios were 0.010, 0.015, 0.015 for control, CIH, and CCH at P30, respectively. The decrease in liver, kidney, and brain weight were greater in CCH than CIH. Smaller liver weight was shown to be due to a reduction in cell size and cell number. Liver in CIH and CCH mice showed a 5% and 10% reduction in cell size (P < 0.05) and a reduction of 28% in cell number (P < 0.001) at P30. In contrast, CCH and CIH heart weight was 13% and 33% greater than control at P30 (P < 0.05), respectively. This increase in the heart weight was due to an increase in the size of cardiomyocytes which showed an increase of 12% and 14% (P < 0.001) for CIH and CCH, respectively as compared to control. Brain weight was 0.48 and 0.46 g for CIH and CCH, respectively (95% and 92% of normal). We concluded that (a) CIH and CCH follow different body and organ growth patterns; (b) mostly with CCH, the liver and kidneys are reduced in size in a proportionate way to body size but heart, lung, and brain are either spared or increased in size compared to body weight; and (c) the decrease in liver is secondary mostly to a decrease in cell number. Pediatr Pulmonol. 2008; 43:20–28. ß 2007 Wiley-Liss, Inc. Key words: hypoxia; postnatal; growth; development; organ; weight; cell size. 1 Department of Pediatrics, New York Medical College, Valhalla, New York. 2 Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York. 3 Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York. Grant sponsor: National Institute of Health; Grant numbers: R01-HL66327, PO1 HD 32573. *Correspondence to: Gabriel G. Haddad, MD, Department of Pediatrics, University of California, 9500 Gilman Drive, La Jolla, CA 92093. E-mail: ghaddad@ucsd.edu Received 8 May 2007; Revised 11 July 2007; Accepted 6 August 2007. 4 Department of Pediatrics, University of California, San Diego, California. 5 Department of Pathology, Albert Einstein College of Medicine, Bronx, New York. 6 The Rady Children’s Hospital, San Diego, California. ß 2007 Wiley-Liss, Inc. DOI 10.1002/ppul.20729 Published online in Wiley InterScience (www.interscience.wiley.com). Effect of Hypoxia on Postnatal Growth 21 INTRODUCTION Hypoxia Exposure Different paradigms of oxygen deprivation can occur in various conditions and disease states. Chronic constant hypoxia (CCH), for example, occurs in disease states such as in congenital heart disease (CHD) and bronchopulmonary dysplasia (BPD). Chronic intermittent hypoxia (CIH) is present in obstructive sleep apnea (OSA), sickle cell anemia and asthma.1–7 These various paradigms of hypoxia often result in different consequences on growth and development. Hypoxia leads to many consequences but one that affects individual subjects early in life is growth and development causing growth deficits or cognitive and behavioral abnormalities.4,8–14 For example, hypoxia in the prenatal period can lead to in-utero growth retardation resulting in low birth weight.15–17 Rodents or humans born or raised at high altitude show a reduction in body weight.18–22 The interference of hypoxia with growth and development4,23–26 depends on the severity and duration of hypoxia and age at the time of exposure. Extensive clinical and experimental data have supported this conclusion at an organismal as well as at a cellular level.15,27,28 Furthermore, we and others have shown that hypoxia affects cell fate and function.29–34 Hypoxia-induced gene regulation is rather well studied and such regulation can have long lasting effects.29–38 There is a large body of literature on the effects of hypoxia on growth and that O2 deprivation reduces body weight in animals in early life.15,28,29 However, most of these studies have focused on a particular organ or they have not compared CCH and CIH as comprehensively as in the present study even though they suspect that CIH imposes oxidative stress in addition to its hypoxic effect. Further, little is known about maternal exposure and litter size. Therefore, in this study, we hypothesize that different hypoxia paradigms, that is, CIH and CCH, as different types of stress, have different effects on growth. We exposed newborn mice to delineate the consequence of long-term hypoxia on body growth and development of specific organs. Our data show that CIH and CCH have different effects on body and organ growth. These data provide an initial and important step to examine effect of CIH and CCH on early postnatal growth. Mice were exposed to hypoxia as previously described.35 Briefly, P2 pups of mixed gender were housed with their own birth dams in isobaric chambers. A combination of nitrogen (N2) and O2 was injected into the chambers to achieve a final concentration of 11% oxygen. For CCH, the O2 level was maintained at 11%
0.1 constantly. For CIH, every cycle consisted of a 4 min period during which O2 level was maintained at 11%
0.1 followed by a 4 min period at 21%
0.1. The ramp time between the two levels took < 11=2 min. This cycle was repeated throughout the hypoxia exposure experiments. The flow of gases into the chambers was controlled by an Oxycycler (Model A44x0, BioSpherix, Refiled, NY) which, in turn, was controlled by ANA-Win2 Software (Version 2.4.17 developed by Watlow Anafaze, Watsonville, CA). The concentrations of O2 and CO2 were monitored by specific electrodes. A feedback system from these electrodes to the controller continuously adjusted the opening of a set of solenoid valves that controlled the flow rate of each gas and hence dynamically maintained a desired gas mixture in the chamber. The level of ambient CO2 was kept at <0.1% via a flow-through system during experiments. Ambient temperature and humidity of gases were monitored and maintained at 22– 248C and 40–50%, respectively. Normal control mice were kept in the same room and were exposed to the same level of noise and light. METHODS Effect of Litter Size Animals Litters with four or eight pups were raised in hypoxia with their own lactating dams. Control litters with the same number of pups (4 or 8) were raised in room air as described above. Pregnant CD1 mice were obtained from Charles River Laboratories (Raleigh, NC). CD1 mice have been used to facilitate comparison with data obtained previously from our laboratory with this strain. This study including animal husbandry and surgery was reviewed and approved by Albert Einstein College of Medicine Institutional Animal Care and Use Committee (IACUC). Animal Litters Used There were a total of eight litters for control and CIH cohorts and nine litters for CCH cohort. Litters were culled to eight pups and two pups from each litter were used at every time point (P9, 16, 23, and 30). For body weight, the number of pups used was 64, 47, 32, 16 for control; 62, 48, 32, 10 for CIH; and 72, 53, 35, 16 for CCH at each time point. For brain, heart, liver and kidney weights at the same ages, the number of pups used was 17, 15, 16, 16 for control, 14, 16, 16, 10 for CIH and 19, 17, 17, 15 for CCH. Number of animals used at each time point is summarized in Table 1. Pups were weaned at P30 and this delay in weaning from a usual of 21 to 30 days was done with the approval of the Albert Einstein College of Medicine Animal Institute Committee. Rotational Studies In order not to subject the lactating dam to hypoxia, we developed a rotating strategy. One litter with eight pups 22 Farahani et al. TABLE 1— Body and Organ Weight Weight Body Control CIH CCH Lung Control CIH CCH Heart Control CIH CCH Brain Control CIH CCH Liver Control CIH CCH Kidneys Control CIH CCH Body/organ weight P2 P9 P16 P23 P30 1.96 (n ¼ 64) 1.87 (n ¼ 64) 1.82 (n ¼ 72) 7.11 (n ¼ 64) 4.94 (n ¼ 62) 5.11 (n ¼ 72) 12.56 (n ¼ 47) 8.48 (n ¼ 48) 8.26 (n ¼ 53) 19.94 (n ¼ 32) 14.40 (n ¼ 32) 12.76 (n ¼ 35) 28.13 (n ¼ 16) 23.71 (n ¼ 10) 19.78 (n ¼ 16) 0.14 (n ¼ 17) 0.14 (n ¼ 14) 0.11 (n ¼ 19) 0.25 (n ¼ 15) 0.22 (n ¼ 16) 0.20 (n ¼ 17) 0.24 (n ¼ 16) 0.24 (n ¼ 16) 0.20 (n ¼ 17) 0.28 (n ¼ 16) 0.36 (n ¼ 10) 0.30 (n ¼ 15) 0.010 0.015 0.015 0.04 (n ¼ 17) 0.04 (n ¼ 14) 0.05 (n ¼ 19) 0.09 (n ¼ 15) 0.08 (n ¼ 16) 0.12 (n ¼ 17) 0.12 (n ¼ 16) 0.11 (n ¼ 16) 0.15 (n ¼ 17) 0.15 (n ¼ 16) 0.18 (n ¼ 10) 0.20 (n ¼ 15) 0.005 0.007 0.010 0.34 (n ¼ 17) 0.29 (n ¼ 14) 0.28 (n ¼ 19) 0.45 (n ¼ 15) 0.41 (n ¼ 16) 0.38 (n ¼ 17) 0.48 (n ¼ 16) 0.44 (n ¼ 16) 0.44 (n ¼ 17) 0.51 (n ¼ 16) 0.48 (n ¼ 10) 0.46 (n ¼ 15) 0.018 0.020 0.023 0.10 (n ¼ 17) 0.09 (n ¼ 14) 0.07 (n ¼ 19) 0.20 (n ¼ 15) 0.18 (n ¼ 16) 0.16 (n ¼ 17) 0.28 (n ¼ 16) 0.23 (n ¼ 16) 0.19 (n ¼ 17) 0.45 (n ¼ 16) 0.42 (n ¼ 10) 0.31 (n ¼ 15) 0.057 0.066 0.059 0.10 (n ¼ 17) 0.09 (n ¼ 14) 0.07 (n ¼ 19) 0.20 (n ¼ 15) 0.18 (n ¼ 16) 0.16 (n ¼ 17) 0.28 (n ¼ 16) 0.23 (n ¼ 16) 0.19 (n ¼ 17) 0.45 (n ¼ 16) 0.42 (n ¼ 10) 0.31 (n ¼ 15) 0.016 0.017 0.015 Average body and organ weight (in grams) for control, CIH, and CCH mice at all the experimental time points are summarized (n is the number of animal used in each time point). Last column on the right shows organ/body ratios at P30. These values are shown in three decimal points for finer accuracy. was used in each group (normoxic, hypoxic, rotating in normoxic, rotating in hypoxia). Six additional litters kept in normoxia, provided lactating dams every day for the rotational hypoxia experiments. The rotating experiments were performed only on the CCH (not the CIH groups) as the CCH animals were in general more affected in body weight than the CIH ones. Data Collection and Statistics In order to avoid frequent room air exposure, hypoxic mice were weighed once a week. Tissue and organs were collected at P9, P16, P23, and P30 (corresponding to 7, 14, 21, and 28 days of hypoxia exposure). For tissue collection, mice were deeply anesthetized using isoflurane (Baxter, Deerfield, IL) immediately prior to organ collection. Both atria, ventricles and vessels were included in the heart weight. The spinal cord was not included in the brain. The gall bladder was not removed prior to liver weighing. Both kidneys were included in the weight. Data are reported as means
standard error. Data were analyzed using two-way ANOVA (GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, CA). Differences in the means were considered statistically significant when P < 0.05. Cell Size and Cell Number To determine whether differences in organ size were related to cell size or cell number, animals were perfused with 0.9% NaCl in 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde in 0.1 M PB. Organs were then collected and fixed in 4% paraformaldehyde in 0.1 M PB overnight and transferred to 70% ethanol. Organs were paraffin-embedded, sectioned and stained with hematoxylin and eosin (H&E) using standard protocols. Photomicrographs of liver median lobes were taken at 40. Each photomicrograph was overlaid with a grid and five cells (four corners and center) were picked where the grid intersected. Areas of the hepatocytes were measured using AxioVision version 4.1.1.0 (Carl Zeiss, Thornwood, NY) which can calculate the area of a cell when it is marked. A total of 30 cells in randomly chosen fields from six animals (n ¼ 6) were measured for each cohort. For cell count, all the cells in six randomly chosen fields were counted, a total of 2,558, 3,408, and 3,465 cells for CCH, CIH, and control, respectively. Heart sections of the left and right ventricles were prepared by sectioning from the base to the apex. Micrographs were taken at 63. The diameter of each cardiomyocyte was measured using AxioVision version Effect of Hypoxia on Postnatal Growth 4.1.1.0 (Carl Zeiss) as explained above. A total of 30 cells were measured from six photomicrographs from six animals in each cohort (n ¼ 6). Each group (control, CIH, or CCH) was analyzed in the same fashion as described for liver. We did not make any assumption about cell area. RESULTS Hypoxia Induces Growth Retardation To evaluate the effect of hypoxia on growth, litters of eight pups each were subjected to CIH or CCH with their own dams beginning at P2. Weight of mice in control, CIH, and CCH groups between P2 and P30 is summarized in Table 1. Regression analysis showed that weight increased with age with r2 values of 0.99, 0.97, and 0.94 for control, CCH, and CIH, respectively. Starting at P9 however, mice subjected to hypoxia had a significantly smaller average body weight than mice raised in normoxia (Table 1 and Fig. 1). Between days 2 and 30, slopes were 0.93
0.057, 0.76
0.108, and 0.63
0.061 (g/day, means
SEM) for control, CIH, and CCH, respectively and all pair wise slopes were significantly different from each other (P < 0.001). We further noticed that the CCH and CIH litter groups have different slopes early on versus later during the 4 weeks of hypoxia exposure. Indeed, the slopes between P2 and P16 were 0.46
0.002 and 0.47
0.019 for CCH and CIH, respectively, and these were significantly decreased (almost half) from those of control (0.78
0.012). From P16 to 30, slopes were 1.12
0.033, 1.09
0.143, and 0.82
0.08 for control, CIH, and CCH, respectively, but these were not signifi- Fig. 1. Hypoxia induced body growth retardation. Hypoxia during the first 4 weeks of life induces significant body growth retardation. Average body weight of eight litters (n ¼ 64) raised in normoxia (21% oxygen, open columns), CIH (11% oxygen, gray columns), or CCH (11% oxygen, solid black columns) is shown in grams between P2–P30 (0–28 days of hypoxia exposure). Bars represent standard errors. Asterisks indicate statistical significance when means in CIH and CCH litters were compared to normoxic control (*P < 0.05). # Indicates statistical differences between CIH and CCH. Photograph shows control, CIH, and CCH mice at P30. Notice smaller body in hypoxic mice as compared to control. 23 cantly different from each other, suggesting that a catchup growth has occurred in the latter part of the hypoxic period. Nevertheless, slower weight gain resulted in a 12% (23.71 g) and 23% (19.78 g) lower body weight in CIH and CCH mice (P < 0.001) by P30 as compared to normal mice (28.13 g; Table 1). Hypoxia Induces a Reduction in Organ Weight Lung, heart, brain, kidneys, and liver obtained from all of the pups were weighed at P9, P16, P23, and P30 (Table 1). The ratio of lung/body weight was comparable to control in the first 2 weeks but reached a significantly higher level at P23 and P30 for CIH and only at P30 for CCH (i.e., 0.015, 0.015, and 0.010, respectively; Table 1). Mean heart weights from CCH mice were significantly larger than control at all ages but this was not the case with CIH (Table 1). At P30, CCH and CIH heart weight was 13% (0.18 g on average) and 33% (0.20 g on average) larger than control (0.15 g on average; P < 0.05). Because of the body weight difference for CIH and CCH mice, mean heart/body weight ratios were much larger for CIH and CCH as compare to control at P30 (P < 0.001). Mean heart/body weight ratios at P30 were 0.005, 0.007, and 0.010 for control, CIH, and CCH, respectively (Table 1 and Fig. 2). Indeed, at some ages, the heart/body weight ratios were nearly double or greater (e.g., at P16) than those of normoxia controls (Fig. 2). Average brain weight in CIH or CCH was slightly but significantly smaller than in controls at all ages (Table 1). Because of a relatively smaller reduction in average brain weight compared to body weight, the ratio of brain/body weight in CIH and CCH were higher than control (0.020, 0.023, and 0.018, respectively at P30), indicating that brain growth was relatively spared compared to body weight (Table 1 and Fig. 2). The liver from CIH and CCH mice showed an interesting growth pattern. Both CIH and CCH mice had livers with significantly reduced weights in the first 2 weeks but showed catch-up growth in subsequent weeks, more robustly in CIH than CCH (Table 1). In fact, CCH mice had an average liver weight significantly smaller at all ages (0.31 vs. 0.45 g at P30). Average liver/body weight ratios were significantly lower early on, especially by P16, but became more comparable to control at later ages, that is, 0.057, 0.066, and 0.059 for control, CIH, and CCH at P30, respectively (Table 1 and Fig. 2). Although average kidney weights in CCH mice were less than controls at all ages (Table 1), significant differences from controls were present only after the first 2 weeks of exposure (at P23, P < 0.01, and at P30, P < 0.001). CIH mice had a tendency for smaller kidneys but significance was achieved only at P23 (Table 1). There were no significant differences in either CIH or CCH for kidney/body weight ratios as compared to normal kidneys 24 Farahani et al. (0.017, 0.015 and 0.016; Table 1), suggesting a remarkable commensurate decrease in kidney size as in body size (Fig. 2). Maternal Effect on Postnatal Growth in Hypoxia In order to determine whether a hypoxic dam (present in chamber with pups) and its nutrition affected pup growth, we rotated dams from other litters (raised in normoxia) into the hypoxic chamber every day so that the dam of the litter raised in hypoxia is present in the chamber only one day per week. This strategy did not improve the weight gain in the CCH pups raised with their own dam (Fig. 3A). At P23, CCH litters with their own dams continuously in hypoxia did not have significantly different weights from those who had rotating foster dams in hypoxia (average body weight of 8.23 and 8.56 g, respectively). Also, litters with rotating dams in normoxia did not have significantly different weights than those raised with their dams in normoxia (17.25 and 17.79 g, respectively). However, litters with rotating foster dams in hypoxia showed significantly lower mean body weight as compared to their controls, kept in normoxia with the rotating dams (8.56 and 17.79 g, respectively). Also mice in CCH with their own dam showed a significant difference in body weight as compared to their control, that is, mice in normoxia with their own dam at all times (8.23 and 17.25 g, respectively). Effect of Litter Size on Postnatal Growth To assess whether litter size had an effect on growth during hypoxia, we exposed litters of four or eight pups to CCH with the same size litters raised in normoxic conditions (Fig. 3B). Litters of four pups gained slightly more weight, on average, when compared to litters of eight pups, whether in normoxia or hypoxia (27.27 and 28.5 g or 4.4% for normal litters and 16.76 and 18.05 g or 7.2% for CCH litters). Litters of four and eight pups raised in hypoxia gained significantly less weight as compared to their respective controls by P30 (18.05 and 28.5 g for CCH and control of four pups, respectively or a 36% difference and 16.76 and 27.27 g for litters of eight pups, respectively, or a 41% difference). Results of these experiments confirmed that hypoxic animals will have significantly lower body weights at P30 whether litter size is progressively reduced at each time point or all the pups are kept and weighed at P30. Fig. 2. Hypoxia induced differential organ growth. Lung, heart, brain, kidneys, and liver were collected from mice raised in normoxia, CIH or CCH at P9, P16, P23, and P30 and weighed. Ratio of each organ to body weight was calculated. A: The ratio of lung/body weight was comparable to control in the first 2 weeks but reached a significantly higher level at P23 and P30 for CIH and only at P30 for CCH (i.e., 0.015, 0.015, and 0.010, respectively). B: There were significant differences at all time points between CCH and normoxic heart/body weight ratios (*P < 0.001). C: Brain/ body weight ratios were higher in hypoxic animals at all time points. Differences were statistically significant at P9, P16, and P23 (*P < 0.001) for CIH and at P9, P16, P23, and P30 (*P < 0.001) for CCH. D: Liver/body weight ratio was significantly lower at P16 (*P < 0.05 and *P < 0.01 for CIH and CCH respectively). E: Kidney/ body weight ratios in hypoxic mice as compared to normoxic mice did not reach any statistical significance. Bars represent standard errors. Asterisks indicate statistical significance when means in CIH and CCH litters were compared to normoxic control. Effect of Hypoxia on Postnatal Growth 25 (P < 0.05), that is, 144.04 and 135.26 mm, respectively as compared to control which was 150.10 mm (Fig. 4 Graph B). Hence, the smaller livers from animals raised in hypoxia resulted from both a decrease in cell number and a decrease in cell size. Examination of cardiomyocytes from left and the right ventricles revealed an interesting pattern in hypoxic hearts, as compared to control (Fig. 4 Graphs C,D). Cardiomyocytes in the left ventricle were significantly larger (approximately 13.5%, P < 0.001) in CCH (75.43 mm) than in control or CIH (57.23 and 55.6 mm, respectively). In the right ventricle, both CIH and CCH cardiomyocytes were significantly larger than control (74.02, 84.19, and 58.05 mm, respectively or 12% and 14% larger, P < 0.001). CIH cardiomyocytes were also significantly smaller than those of CCH (P < 0.05). Fig. 3. Maternal and litter size effect on postnatal growth. Panel A: Litters of eight pups were raised in CCH (black columns) or normoxia (white columns) for 3 weeks starting at P2 either with their own dams at all times or with rotating foster dams (hatched columns). Average weight of each litter at P23 (after 3 weeks of exposure to CCH) is shown. Average weight of normoxic litter kept with their birth dam at all times was comparable to that of the litter with rotating dams in normoxia. Also, average weight of CCH litter kept with their birth dam at all times was comparable to that of the CCH litter with rotating dams. However, average weights of litters kept in hypoxia (whether kept with their birth dam at all times or with foster dams) were comparable to each other and significantly smaller than their respective control litters raised in normoxia (*P < 0.001). Panel B: Litters of four or eight pups were raised in normoxia as control (white columns) or in CCH (black columns) for 4 weeks. Average weight of each litter at P30 is shown. Average weight of litters (with 4 or 8 pups) kept in CCH was significantly smaller (*P < 0.001) as compared to the average weight of their respective controls (litters with four or eight pups). Average weight of litters with four or eight pups was not significantly different whether raised in normoxia or hypoxia. Bars represent standard errors. Asterisks indicate statistical significance when means in CIH and CCH litters were compared to normoxic control (*P < 0.05). Cell Size and Cell Number As shown above, growth rate during hypoxia varied from organ to organ. We therefore asked whether the change in organ size is mostly related to a change in cell size or cell number. Cell size and cell number were determined for heart and liver as the prototype of organs which showed different growth patterns in response to hypoxia; one increasing and the other decreasing in size. Sections from liver and heart, from both CIH and CCH at P30, were examined. The number of CIH and CCH hepatocytes per area (426.33 and 426.33 cells/area, respectively) were approximately 28% lower (P < 0.001) as compared to control which was 577.50 cells/area (Fig. 4 Graph A) at P30. Moreover, area of hepatocytes from CIH or CCH were on average 5–10% smaller than control hepatocytes DISCUSSION This study was undertaken to investigate the growth pattern of mice exposed to two different types of hypoxia, that is, CIH and CCH. We have made several interesting observations: (1) the growth pattern of mice in CIH is different from that in CCH; (2) there are two patterns of organ growth that are rather different in each of the two hypoxic paradigms; (3) the pattern of growth for certain organs is different in CIH as compared to CCH; (4) rotating mothers and minimizing the exposure time for dams to hypoxia, or reducing the number of pups exposed to hypoxia, did not alleviate growth inhibition in hypoxia; and (5) the hypoxia-induced reduction in organ size, such as for the liver, is based mostly on cell number, although cell size is also decreased, especially in CCH. It has been shown by a number of investigators that hypoxia retards body growth.21,27,28 These studies have been mostly done in rats and only in CCH. In this investigation, we have compared mice exposed to CCH with those exposed to CIH. We found that mice exposed to CIH have a rather similar pattern of growth retardation in the first 2 weeks of exposure as CCH mice but that they differ from CCH in the latter 2 weeks, since mice exposed to CIH show a brisk catch-up growth between P16 and P30 while those exposed to CCH do not show as much. The difference in CIH and CCH are present in the last 2 weeks but reaches significance at P30. It seems from our studies that there are two distinct growth patterns for organs in the hypoxia paradigms used. The first, exemplified by the heart, especially in CCH, is that of an enhanced weight and growth. The second, exemplified by the liver and kidney, is that of a reduced weight and size. In CCH, it was anticipated that heart size increases, as there are numerous reports, including our own,29 showing cardiac hypertrophy when subjects are exposed to constant hypoxia for days and weeks.39,40 26 Farahani et al. Indeed, not only did we previously show29 that hypertrophy occurs in CCH but we also noticed a number of interesting findings related to translation and transcriptome of a number of genes such as the initiation and elongation factors (eI2 and eI4 mRNA and protein). From a functional point of view, this hypertrophy is useful in increasing cardiac output and oxygen delivery. A decrease in liver and kidney weights paralleled with body weight in such a way that the ratios of organ/body weights were similar in CIH, CCH, and control. Since one could argue that renal mass and size as well as renal function are tied to bodily metabolic needs, it may not be surprising that kidney to body ratio is constant, even during hypoxia. A similar argument could be entertained for the liver as well. In this study we have assessed differences in the organ growth using morphological and histological techniques to measure cell size and cell number. These approaches may not provide data regarding total DNA/protein. The brain is somewhat smaller in CCH but this reduction is much smaller than that of body size, implying sparing of brain mass, even when the stress occurs in early life, and when CCH is rather severe. Clearly, a relative sparing of brain does not necessarily indicate a lack of dysfunction, as has been documented by other researchers.41,42 In fact, we have recently reported on dysmyelination and, more importantly, that this abnormal myelination was not reversible after reoxygenation.35 It is also unknown if there is a difference in brain function after CIH as compared to CCH. Gozal and colleagues have argued in the past that CIH is more deleterious in terms of brain function since there is evidence of apoptosis and memory dysfunction after CIH as compared to CCH.42–44 It is possible, therefore, that CIH affects brain function more than CCH since CIH is tantamount not only to periodic decreases in O2 but also intermittent increases in O2 which may act as ‘‘periodic oxidants.’’34,45 One of the important issues in this work relates to the genesis of body and organ size reduction. Our data, which show that this reduction is not alleviated by culling the exposed litter from eight to four pups or by rotating mothers in the exposure chamber, strongly suggest that (a) Fig. 4. Hypoxia differentially affects cell size and cell number in liver and heart. Plots in panels A,B show liver cell number or cell size for control (white column), CIH (gray column) or CCH (black column), respectively. Both CIH and CCH livers have a significantly smaller number of cells (*P < 0.001). Interestingly, CCH but not CIH hepatocytes are significantly smaller in size (*P < 0.05) as compared to control hepatocytes (144.04, 135.26, and 150.10 mm, respectively) which represented 5% and 10% reduction in cell size (P < 0.05) and 28% in cell number (P < 0.001) at P30 (see Results Section for details). Plots in panels C,D show left (LV) and right (RV) ventricle cell size for control (white column), CIH (gray column) or CCH (black column), respectively. CCH but not CIH hearts have significantly larger cells in the left ventricle (*P < 0.001). Interestingly, right ventricular cardiomyocytes were significantly larger in both CIH and CCH hearts with CCH having larger cells as compared to both control and CIH (*P < 0.001 and *P < 0.001, respectively). Asterisks indicate statistical significance when means from hypoxic animals were compared to normoxic controls. Bars represent standard errors. Effect of Hypoxia on Postnatal Growth maternal nutritional effects may not play a major role and (b) it is the hypoxic stress that induces this retarded growth. Evidence in the literature exists to support this contention. One of the latest studies on the subject of hypoxia and nutritional/maternal factors, performed by Mortola et al.,28 concluded that neonatal growth retardation during moderate (15% O2) or severe (10% O2) hypoxic exposure can be almost entirely attributed to hypoxia, and is not mediated by maternal responses. A number of other studies have also been done at high altitude on children in the Andes, Ethiopian highlands, and Asian Himalayas. Although this same issue has been raised, nutrition versus hypoxia per se, it is becoming clear that (i) there is growth retardation, including at birth, of children born at high altitude; and (ii) when controlled for nutrition, socio-economic class and other factors, studies have concluded that growth and development at high altitude result in a moderate delay in linear growth of wellnourished children, and that these patterns are established very early in life.46,47 One very interesting observation that has bearing on this issue is related to hypoxia in invertebrates.48,49 Fruit flies (Drosophila) decrease their body size as a function of severity of the hypoxic stress.48,49 Cell numbers and cell size in flies reared in 5% oxygen decreased by 25–30% as compared to flies that live in normoxia.48 Hence, invertebrates, such as flies, seem to behave under hypoxic conditions in a similar way to mice and humans in relation to body size. Organs, like liver and kidney, which decrease in size with hypoxia, seem to have a lower number of cells, as shown in Figure 4. Hepatocyte size may also be affected, albeit to a lesser degree, and mostly in CCH. While the mechanisms for this reduction in cell number or size are not clear, we have shown previously that hypoxia can prolong cell cycle in Drosophila or even halt the cycle completely.50 Such a slowing, for example, will result in slower cell division and a reduction in cell number per organ or tissue. Litters of four pups gained slightly more weight, on average, when compared to litters of eight pups, whether in normoxia or hypoxia (4.4% for normal litters and 7.2% for CCH litters). Litters of 4 and 8 pups raised in hypoxia gained significantly less weight as compared to control by P28 (36% and 41%, respectively). Moreover, results of these experiments confirmed that the hypoxic animal will have a significantly lower body weight at P30 whether litters size is progressively reduced at each time point or all the pups are kept and weighed at P30. These studies strongly suggest that hypoxia per se has an effect on the growth of pups. In summary, we have studied the growth of mice and selected organs (heart, lung, brain, liver, and kidneys) over the first month of life when subjected to either chronic constant or cyclical hypoxia. Rotating dams (in order not to subject lactating dams to chronic hypoxia) or reducing 27 the number of pups by half did not have any effect on the decrease in the weight of these mice. Organ growth, represented by heart and liver which illustrated two patterns of growth in mice exposed to constant hypoxia, was either commensurate with bodily growth or was spared or increased in actual size. ACKNOWLEDGMENTS We are grateful to Cate Muenker and Adrianna L. 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